allb-beta3 and aVbeta3 integrins mediate adhesion of platelets and other cells to matrix ligands at sites of vascular injury. Ligand binding is regulated by activating or "inside-out" signals that modulate integrin affinity through conformational changes and integrin avidity through microclustering. Recently, integrin conformational changes have been characterized structurally and talin has been identified as a key regulator of these changes by binding to the beta3 cytoplasmic tail. Here, three major unresolved questions will be asked concerning the molecular basis of inside-out signaling. First, what is the relationship between talin and two other proteins, Rap1b and ADAP, recently implicated as positive regulators of beta3 integrin affinity? The role of Rap1b or ADAP in talin-dependent beta3 integrin activation will be assessed in primary mouse megakaryocytes, platelets and an improved CHO cell model system in which beta3 integrins are sensitive to inside-out signals. Over-expression and knockdown approaches will be used to investigate the inter-dependency of these mediators and their relative effects on beta3 integrin affinity and microclustering. Then, Rap1b and ADAP effectors that regulate beta3 integrins will be identified and their actions characterized. Second, are there regulators of beta3 integrin affinity still to be discovered? Here, the effects on allb-beta3 affinity of PTB domain- containing proteins other than talin will be determined, focusing particular attention on moesin and Dok-2, platelet proteins implicated in cytoskeletal regulation. In a complementary approach, affinity regulation of beta3 integrins will be characterized in zebrafish to evaluate mechanistic similarities and differences with the regulatory process in mammals. Third, what are the mechanisms and consequences of beta3 integrin activation in neoplastic cells? Over-expression and knockdown approaches will investigate whether Rap1b or talin are required for constitutive aVbeta3 activation in certain tumor cells and whether this integrin activation promotes a metastatic phenotype. The potential for oncogenic activation of allb-beta3 in chronic myeloproliferative diseases will be evaluated in mouse megakaryocytes and platelets by over-expressing constitutively-active JAK2 (V617F), a common mutation in these bleeding- and thrombosis-prone diseases. Together, these studies will provide insights into how beta3 integrin activation is regulated, how the activation mechanism is co-opted in clinical disorders, and how it may be harnessed to develop better anti-platelet drugs.